[unreadable] Biofilms are complex bacterial communities attached to a surface. The most widely recognized property of biofilm bacteria is their increased resistance to antimicrobial agents. The recalcitrance of biofilm-related infections to conventional antibiotic therapy has a profound impact on the medical industry and human health. Although this problem has been studied in numerous different model systems, little is known about the molecular mechanisms that confer antibiotic resistance to biofilm cells. Recent work suggests that a genetic program controls biofilm formation and we have genetic evidence that indicates that the development of antibiotic resistance in biofilms is similarly regulated. Furthermore, so-called "small colony variants" or SCV have been proposed to play a role in biofilm-mediated resistance in P. aeruginosa. The role of SCV in S. aureus biofilm resistance has not yet been investigated. The central hypothesis of this application is that antimicrobial resistance of biofilm-grown cells requires specific genetic elements. We propose to identify genetic elements required for the development of biofilm antibiotic resistance by Staphylococcus aureus using techniques developed in our previous studies in Pseudomonas aeruginosa. The identification of genes required for biofilm antibiotic resistance may provide new targets for anti-biofilm therapies and increase our understanding of biofilm antibiotic resistance. [unreadable] The Specific Aims of this application are: [unreadable] Specific Aim 1. Identify genetic elements required for biofilm antibiotic resistance in S. aureus. [unreadable] Specific Aim 2. Characterize mutants defective in biofilm antibiotic resistance. [unreadable] Specific Aim 3. Determine the role of small colony variants (SCV) in biofilm antibiotic resistance. [unreadable] The studies proposed here explore a poorly characterized aspect of microbial resistance that is elaborated when microbes grow in a biofilm. No genetic elements contributing to biofilm resistance have been identified in S. aureus. We propose a genetic screen (already validated in P. aeruginosa) with the goal of identifying genes involved in this process. Future studies will uncover the mechanisms of resistance mediated by the genes identified as a result of this work. We will also perform studies to determine whether SCV, thought to be generated in chronic bacterial infections in vivo: i) play a role in biofilm resistance and ii) utilize the same genetic pathways as biofilm-grown cells to resist the action of antibiotics. [unreadable] [unreadable]